Field of the Invention
The present invention relates to systems and methods for performing multiple diagnostic assays simultaneously, and more particularly to systems and methods for distinguishing different measured emission signals based on the modulation frequencies of the corresponding excitation signals that generated the emission signals.
Background of the Invention
None of the references described or referred to herein are admitted to be prior art to the claimed invention.
Diagnostic assays are widely used in clinical diagnosis and health science research to detect or quantify the presence or amount of biological antigens, cell or genetic abnormalities, disease states, and disease-associated pathogens or genetic mutations in an organism or biological sample. Where a diagnostic assay permits quantification, practitioners may be better able to calculate the extent of infection or disease and to determine the state of a disease over time. Diagnostic assays are frequently focused on the detection of chemicals, proteins or polysaccharides antigens, nucleic acids, biopolymers, cells, or tissue of interest. A variety of assays may be employed to detect these diagnostic indicators.
Nucleic acid-based assays, in particular, generally include multiple steps leading to the detection or quantification of one or more target nucleic acid sequences in a sample. The targeted nucleic acid sequences are often specific to an identifiable group of proteins, cells, tissues, organisms, or viruses, where the group is defined by at least one shared sequence of nucleic acid that is common to members of the group and is specific to that group in the sample being assayed. A variety of nucleic acid-based detection methods are fully described by Kohne, U.S. Pat. No. 4,851,330, and Hogan, U.S. Pat. No. 5,541,308.
Detection of a targeted nucleic acid sequence frequently requires the use of a nucleic acid molecule having a nucleotide base sequence that is substantially complementary to at least a portion of the targeted sequence or its amplicon. Under selective assay conditions, the probe will hybridize to the targeted sequence or its amplicon in a manner permitting a practitioner to detect the presence of the targeted sequence in a sample. Techniques of effective probe preparation are known in the art. In general, however, effective probes are designed to prevent non-specific hybridization with itself or any nucleic acid molecule that will interfere with detecting the presence of the targeted sequence. Probes may include, for example, a label capable of detection, where the label is, for example, a radiolabel, a fluorophore or fluorescent dye, biotin, an enzyme, a chemiluminescent compound, or another type of detectable signal known in the art.
Because the probe hybridizes to the targeted sequence or its amplicon in a manner permitting detection of a signal indicating the presence of the targeted sequence in a sample, the strength of the signal is proportional to the amount of target sequence or its amplicon that is present. Accordingly, by periodically measuring, during an amplification process, a signal indicative of the presence of amplicon, the growth of amplicon overtime can be detected. Based on the data collected during this “real-time” monitoring of the amplification process, the amount of the target nucleic acid that was originally in the sample can be ascertained. Systems and methods for real time detection and for processing real time data to ascertain nucleic acid levels are described, for example, in Lair, et al., U.S. Pat. No. 7,932,081, “Signal measuring system for conducting real-time amplification assays,” the disclosure of which is hereby incorporated by reference.
To detect different nucleic acids of interest in a single assay, different probes configured to hybridize to different nucleic acids, each of which may provide detectably different signals can be used. For example, different probes configured to hybridize to different targets can be formulated with fluorophores that fluoresce at a predetermined wavelength when exposed to excitation light of a prescribed excitation wavelength. Assays for detecting different target nucleic acids can be performed in parallel by alternately exposing the sample material to different excitation wavelengths and detecting the level of fluorescence at the wavelength of interest corresponding to the probe for each target nucleic acid during the real-time monitoring process. Parallel processing can be performed using different signal detecting devices constructed and arranged to periodically measure signal emissions during the amplification process, and with different signal detecting devices being configured to generate excitation signals of different wavelengths and to measure emission signals of different wavelengths.
Occasionally, however, the excitation and emission wavelengths for one fluorophore will overlap the excitation and emission wavelengths of another fluorophore. In such circumstances, it becomes a challenge to ensure that a measured signal is entirely due to an emission of the fluorophore of interest, excited by an intended excitation signal. Such “optical crosstalk” can take a number of forms. For signal detecting devices configured to measure emissions from samples held in closely adjacent reaction receptacles, crosstalk can occur when one channel detects the excitation light from another channel (of the same or different signal detector) or when one signal detecting device picks up emission light from a receptacle that is excited by a different signal detecting device. In addition, crosstalk can occur when an excitation signal for a particular dye color excites a dye of a color that is not intended for that signal detector. Furthermore, crosstalk can occur when an emission signal for a particular dye color excites a dye of a color that is not intended for that signal detector.
Synchronous detection is a means to reduce some forms of crosstalk, as well as optical noise due to, for example, ambient light. Synchronous detection creates a narrow bandwidth filter that is sensitive to a narrow range of frequencies in the emission signal centered at a modulation frequency of the excitation signal. The excitation signal from the signal detecting device is demodulated at the modulation frequency, and a fluorescence detection circuit is configured to detect the frequency of the measured emission signal and to reject portions of the signal having a frequency that is inconsistent with the excitation frequency. Such a circuit-based “analog demodulator” is described, for example, in Lair, et al., U.S. Pat. No. 7,932,081.